Caspases are an ancient class of cysteinyl proteases that are critical to apoptosis and cell differentiation, but little is known about how caspase activity is modulated for apoptotic versus non- apoptotic events. Our primary goal in this proposal is to determine evolutionary trajectories from a common scaffold that resulted in modulation of activity in extant caspase clusters. The apoptotic caspases evolved from a common ancestor into two distinct subfamilies that are either monomers (initiator caspases) or dimers (effector caspases). Biologically, dimerization of initiator caspases is an important cell-fate determining property; conversely, dimeric effector caspases evolved unique allosteric mechanisms to fine-tune activity. Our long-term goal is to integrate evolutionary biology with rigorous biochemical studies to define the evolutionary trajectories in caspases. These studies will stimulate new areas for evolutionary biochemical studies on apoptotic regulatory mechanisms, protein oligomerization, and developing enzymes with altered specificities. The objective of this grant is to characterize mutations that occurred in the common ancestor that resulted in two distinct subfamilies of apoptotic caspases. The central hypothesis is that limited mutations in the caspase-hemoglobinase scaffold established the folding and conformational landscapes >650 million years ago and that extant caspases evolved different properties through differentially modifying common interaction networks. Our rationale is that studies of reconstructed ancestral proteins suggest that mutations in a weak ancestral dimer resulted in two subfamilies with different oligomeric properties. Neofunctionalization of the two subfamilies provided distinct enzyme substrate selection as well as allosteric mechanisms to modulate activity. Our specific aims will test the following hypotheses: (Aim1) A weakly dimeric common ancestor provided a platform for evolution of dimeric and monomeric caspase subfamilies; (Aim 2) A promiscuous common ancestor provided a platform for minimal modifications that resulted in modern enzyme selection; (Aim 3) Evolutionary changes in a common allosteric network resulted in unique regulatory mechanisms through selection of different conformations in the native ensemble. This contribution is significant since it will establish key features of substrate specificity and allosteric regulation that were retained through hundreds of millions of years of evolution, while other regulatory features are modern and cluster-specific. The proposed research is innovative because we established a database (CaspBase) containing over 6,600 caspase sequences from 353 taxa in order to infer ancestral sequences and reconstruct ancestral proteins, which are used to characterize evolutionary trajectories. Insight into caspase evolution is impactful because properties retained for more than 700 million years in the ancestral scaffold can be used to generate new enzymes with altered specificities and allosteric regulation.